title: Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay.
abstract: DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.
authors.affiliation: Centre for Brain Ageing and Vitality, Human Nutrition Research Centre, Institute for Ageing & Health, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
authors.forename: Joanna P
authors.initials: JP
authors.lastname: Gorniak
authors.forename: Kerry M
authors.initials: KM
authors.lastname: Cameron
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Waldron
authors.forename: Thomas
authors.initials: T
authors.lastname: von Zglinicki
authors.forename: John C
authors.initials: JC
authors.lastname: Mathers
authors.forename: Sabine A S
authors.initials: SA
authors.lastname: Langie
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D003106:Colon / Q000201:enzymology; D020552:Comet Assay / Q000379:methods*; D053837:DNA Cleavage; D004260:DNA Repair; D003851:Deoxyribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity
chemical_list: D016590:Aphidicolin; D003851:Deoxyribonucleases